The focus of this proposal is to develop further potential glycomarkers we have identified for pancreatic cancer and breast cancer, as well as to discover new glycomarkers by detailed glycomic analysis of the tumor initiating cells (TIC) of pancreatic and breast cancer and of tumor-associated stellate cells that surround pancreatic cancer. Clearly, developing biomarkers for pancreatic and breast cancer is of critical importance in the early detection of cancers. During the first cycle of the NCI U01 focused on Glycans and Cancer, we analyzed pancreatic ductal fluid from those with pancreatic ductal adenocarcinoma and controls using glycomic analysis tools developed in our NCRR Resource for Biomedical Glycomics. We have identified several potential glycomarkers that will be developed in this competitive renewal application. Most notably, we have identified a unique N-glycan that shows promise as a sensitive and specific marker of pancreatic adenocarcinoma. In addition, we developed a Targeted Glycoproteomics technology and used it to identify potential markers in invasive ductal breast carcinoma tissue. Two glycoprotein glycomarkers were identified and verified in serum samples. A newly devised sandwich ELISA to detect the specific glycoform of one of these markers shows promise in distinguishing serum from patients with breast cancer and those with benign polycystic disease. In the discovery Aim of this proposal, we will prepare TIC from pancreatic adenocarcinoma by cell sorting and apply glycomics technologies to identify differences between TIC and differentiated tumor cells. A similar strategy will be utilize using cultured breast cancer cell lines. As we have during the first cycle of the U01, we will exploit these differences by devising serum assays to identify potential glycoprotein glycoform markers. Similarly, we will analyze tumor-associated fibroblastic stellate cells from pancreatic cancers and grown in culture for potential glycomarkers. These cells surround these cancers and may, therefore, secrete or release potential glycomarkers for the cancer that can be detected in serum.